New antiviral therapy for influenza is urgently needed to complement vaccination and existing drugs, and to strengthen global efforts to control epidemics and potential new pandemics. New anti-influenza treatments are critical in the face of emerging antiviral resistance to the existing drugs. The long-term objective of this research plan is to develop a safe and highly effective intranasal influenza hemagglutinin (HA)-derived inhibitor as prophylaxis for use in high-risk unvaccinated populations. In preliminary work, we have generated influenza fusion inhibitors by conjugating lipid to specific peptides derived from the C-terminal region of HA and adding cell penetrating peptide sequences for intracellular targeting. We showed that intranasal administration of our lead fusion inhibitor provides antiviral prophylaxis as efficient as the approved drug Relenza(c) in vivo. The fusion inhibitory peptides self-assemble into ~30- 50 nm nanoparticles and are internalized by the target cells. We plan to optimize the candidate antiviral peptides and assess them in vivo to lay the groundwork for human use. To do so, we propose to enhance: 1) antiviral potency; 2) peptide self-assembly; 3) target cell membrane insertion; 4) in vivo biodistribution. We aim to generate candidate peptides that, when administered intranasally, protect the human airway epithelium and prevent viral infection. These goals will be accomplished with two specific aims: 1. Use structure-guided mutagenesis and protein engineering to optimize the antiviral potency and bioavailability of influenza peptide fusion inhibitors. A systematic structural approach will be used to incorporate specific residue substitutions at the inhibitor binding interface that increase the binding energy f the inhibitor to its target. Using a combination of biophysical analysis and bioengineering we will optimize the inhibitors' features -- including particle stability and endosomal localization -- in order to lower the IC50 to nanomolar values. We will conduct in vitro and ex vivo studies to assess the antiviral activity of the engineered inhibitory peptides. 2. Evaluate the protection against influenza infection afforded by optimized self- assembling peptides in cotton rats. We will evaluate the bio-distribution and toxicity properties of the optimized nanoparticles, and assess their in vivo anti-influenza potency. In an iterative process, the outcome of the experiments will guide further optimization, yielding a set of promising investigational anti-influenza agents.